Nucleic acid aptamer capable of detecting ENOX2 protein in serum of cancer patient and application thereof
A nucleic acid aptamer, patient serum technology, applied in the field of biomedical detection and analysis, can solve the problems of insufficient specificity, high production cost, easy to change, etc., and achieves rapid denaturation and renaturation, low production cost, and production cost. high effect
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Embodiment 1
[0102] 1. Synthesize the peptide chain (the peptide chain was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.):
[0103] Ac-EEAKEKFKQALSGILIQFE-Amide: (ie
[0104] Ac-GluGluAlaLysGluLysPheLysGlnAlaLeuSerGlyIleLeuIleGlnPheGlu-Amide) (SEQ ID NO.5)
[0105] 2. Synthesis of a DNA library for screening ENOX2 peptide chain aptamers (the DNA library was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.):
[0106] Apt-L: 5'-GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT (SEQ ID NO. 6), purified by PAGE. Among them, N can be any one of ATGC, a total of 45 N. Therefore, this DNA library should have 445 different nucleotide sequences. From such a large library, aptamers with peptide chain specificity and high affinity to the conserved sequence of ENOX2 were screened out.
[0107] 3. Synthesize three short DNA fragments for PCR primers:
[0108] Apt-F:5'-GGT ATT GAG GGT CGC ATC (SEQ ID NO.8)
[0109] A...
Embodiment 2
[0211] Such as figure 1 As shown, the aptamers SEQ ID NO.1 (Apt-1), SEQ ID NO.2 (Apt-2), SEQ ID NO.3 (Apt -3) and or SEQ ID NO.4 (Apt-4) combined with ENOX2 Western blot; the cDNA of human ENOX2 was cloned into pET-11a expression plasmid, transformed into Escherichia coli, induced by IPGE to express ENOX2 protein, and then extracted for inclusion The total protein in the body, the protein concentration is determined. Take 1 microgram of total inclusion body protein and separate the protein with SDS-PAGE gel according to molecular weight, then transfer the protein to nitrocellulose membrane for WESTERN BLOT analysis. Apt-1, Apt-2, Apt-3 and Apt-4 were labeled with biotin, and the four nucleic acid aptamers labeled with biotin were incubated with nitrocellulose membrane for one hour at room temperature, and after the unbound aptamers were eluted, The membrane was then incubated with streptavidin-alkaline phosphatase, eluted after one hour at room temperature, and the substrate...
Embodiment 3A
[0225] Example 3 Apt-1 inhibits the activity of ENOX2 protein
[0226] The amino acid sequence of human ENOX2 protein is SEQ ID NO.11.
[0227] To determine whether Apt-1 can inhibit the activity of human ENOX2, the present invention uses the effect of the reductase activity of ENOX2 on thioredox protein. To measure thiol production, the turbidity reaction that occurs after mixing insulin and DTT. The result of the present invention shows that after adding the S-tag recombinant human ENOX2 protein, the turbidity is obviously enhanced, indicating that a large amount of thiol is produced. After adding Apt-1 to the reaction, the reductase activity of recombinant human ENOX2 was significantly inhibited. The results are shown in Table 2. It can be seen that Apt-1 can effectively bind human ENOX2 protein, and this specific nucleic acid aptamer can be used to detect ENOX2 protein in serum for early diagnosis of cancer. At the same time, Apt-1 combined with ENOX2 can effectively in...
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